Increased Phosphodiesterase Activity Research Articles

Inhibiting bacterial biofilm formation by stimulating c-di-GMP regulation using citrus peel extract from Jeju Island

Biofilms consist of single or multiple species of bacteria embedded in extracellular polymeric substances (EPSs), which affect the increase in antibiotic resistance by restricting the transport of antibiotics to the bacterial cells. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In this study, we found that citrus peel extract from Jeju Island (CPEJ) can inhibit bacterial biofilm formation. According to the results, CPEJ concentration-dependently reduces biofilm formation without affecting bacterial growth. Additionally, CPEJ decreased the production of extracellular polymeric substances but increased bacterial swarming motility. These results led to the hypothesis that CPEJ can reduce intracellular bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) concentration. The results showed that CPEJ significantly reduced the c-di-GMP level through increased phosphodiesterase activity. Altogether, these findings suggest that CPEJ as a biofilm inhibitor has new potential for pharmacological (e.g. drug and medication) and industrial applications (e.g. ship hulls, water pipes, and membrane processes biofouling control).

The adiponectin agonist, AdipoRon, inhibits steroidogenesis and cell proliferation in human luteinized granulosa cells

During obesity, excess body weight is not only associated with an increased risk of type 2-diabetes, but also several other pathological processes, such as infertility. Adipose tissue is the largest endocrine organ of the body that produces adipokines, including adiponectin. Adiponectin has been reported to control fertility through the hypothalamic–pituitary–gonadal axis, and folliculogenesis in the ovaries. In this study, we focused on a recent adiponectin-like synthetic agonist called AdipoRon, and its action in human luteinized granulosa cells.We demonstrated that AdipoRon activated the adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor alpha (PPAR) signalling pathways in human luteinized granulosa cells. A 25 μM AdipoRon stimulation reduced granulosa cell proliferation by inducing cell cycle arrest in G1, associated with PTEN and p53 pathway activation. In addition, AdipoRon perturbed cell metabolism by decreasing mitochondrial activity and ATP production. In human luteinized granulosa cells, AdipoRon increased phosphodiesterase activity, leading to a drop in cyclic adenosine monophosphate (cAMP) production, aromatase expression and oestrogens secretion.In conclusion, AdipoRon impacted folliculogenesis by altering human luteinized granulosa cell function, via steroid production and cell proliferation. This agonist may have applications for improving ovarian function in metabolic disorders or granulosa cancers.

Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor-mediated quorum sensing.

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor-mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis-2-decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.

Live-Cell Assays for Cell Stress Responses Reveal New Patterns of Cell Signaling Caused by Mutations in Rhodopsin, α-Synuclein and TDP-43.

Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling, we created live-cell assays for endoplasmic reticulum (ER)-mediated cell stress and second messenger signaling. We first examined neurodegenerative mutations associated with direct ER stress by exploring the effect of rhodopsin mutations on ER stress and Ca2+ signaling. The rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa (RP), produced increased ER stress levels compared to wild type (WT) rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca2+ signaling in a stress-dependent manner. Analysis of single-cell Ca2+ signaling profiles revealed unique Ca2+ signaling responses exist in cells expressing WT or P23H rhodopsin, consistent with the idea that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in neurodegenerative diseases that may not have a direct effect on ER-mediated cell stress, we examined how various mutants of α-synuclein and TDP-43 affected ER stress. Mutants of both α-synuclein and TDP-43 associated with Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS) demonstrated increased ER stress compared to WT proteins. To examine the effect of α-synuclein and TDP-43 mutants on cellular signaling, we created a second live-cell assay to monitor changes in cAMP signaling during expression of various forms of α-synuclein and TDP-43. The increased cell stress caused by expression of the mutant proteins was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors for cellular stress and signaling can provide nuanced, new views of neurodegenerative disease processes.

Will you still need me (Ca2+, TnT, and DHPR), will you still cleave me (calpain), when I'm 64?

Will you still need me (Ca²⁺, TnT, and DHPR), will you still cleave me (calpain), when I'm 64?

Modulation of signaling through GPCR-cAMP-PKA pathways by PDE4 depends on stimulus intensity: Possible implications for the pathogenesis of acrodysostosis without hormone resistance

In acrodysostosis without hormone resistance, a disease caused by phosphodiesterase (PDE)-4D mutations, increased PDE activity leads to bone developmental defects but with normal renal responses to PTH. To identify potential mechanisms for these disparate responses, we compared the effect of PDE activity on hormone signaling through the GPCR-Gsα-cAMP-PKA pathway in cells from two lineages, HEK-293 cells stably overexpressing PTH1R (HEKpthr) and human dermal fibroblasts, including studies evaluating cAMP levels using an Epac-based BRET-sensor for cAMP (CAMYEL). For ligand-induced responses inducing strong cAMP accumulation, the inhibition of PDE4 activity resulted in relatively small further increases. In contrast, when ligand-induced cAMP accumulation was of lesser intensity, the inhibition of PDE4 had a more pronounced effect. Similar results were obtained evaluating downstream events (cellular CREB phosphorylation and CRE-luciferase activity). Thus, the ability of PDE4 to modulate signaling through GPCR-cAMP-PKA pathways can depend on the cell type and stimulus intensity.

Role of Extracellular Signal-regulated Kinase 5 in Adipocyte Signaling

Increased adiposity due to energy imbalance is a critical factor of the epidemic crisis of obesity and type II diabetes. In addition to the obvious role in energy storage, regulatory factors are secreted from adipose depots to control appetite and cellular homeostasis. Complex signaling cross-talks within adipocyte are also evident due to the metabolic and immune nature of adipose depots. Here, we uncover a role of extracellular signal-regulated kinase 5 (ERK5) in adipocyte signaling. We find that deletion of ERK5 in adipose depots (adipo-ERK5(-/-)) increases adiposity, in part, due to increased food intake. Dysregulated secretion of adipokines, leptin resistance, and impaired glucose handling are also found in adipo-ERK5(-/-) mice. Mechanistically, we show that ERK5 impinges on transcription factor NFATc4. Decreased phosphorylation at the conserved gate-keeping Ser residues and increased nuclear localization of NFATc4 are found in adipo-ERK5(-/-) mice. We also find attenuated PKA activation in adipo-ERK5(-/-) mice. In response to stimulation of β-adrenergic G-protein-coupled receptor, we find decreased NFATc4 phosphorylation and impaired PKA activation in adipo-ERK5(-/-) mice. Reduced cAMP accumulation and increased phosphodiesterase activity are also found. Together, these results demonstrate integration of ERK5 with NFATc4 nucleo-cytoplasmic shuttling and PKA activation in adipocyte signaling.

NO-Induced Biofilm Dispersion in Pseudomonas aeruginosa Is Mediated by an MHYT Domain-Coupled Phosphodiesterase

Dispersion is a process used by bacteria to successfully transit from a biofilm to a planktonic growth state and to spawn novel communities in new locales. Alterations in bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) levels have been shown to be associated with biofilm dispersal in a number of different bacteria. The signaling molecule nitric oxide (NO) is known to induce biofilm dispersion through stimulation of c-di-GMP-degrading phosphodiesterase (PDE) activity. However, no c-di-GMP modulating enzyme directly involved in NO-induced dispersion has yet been described in the opportunistic pathogen Pseudomonas aeruginosa. Here, we characterized MucR (PA1727) and NbdA (PA3311, NO-induced biofilm dispersion locus A), two membrane-bound proteins with identical domain organization consisting of MHYT-GGDEF-EAL, with respect to their role in NO-induced dispersion. Inactivation of mucR impaired biofilm dispersion in response to NO and glutamate, whereas inactivation of nbdA only impaired biofilm dispersion upon exposure to NO. A specific role of NbdA in NO-induced dispersion was supported by increased PDE activity, resulting in decreased c-di-GMP levels in biofilms expressing nbdA upon exposure to NO, a response that was absent in the ΔnbdA strain. Moreover, increased PDE activity was mainly due to a transcriptional activation of nbdA upon addition of NO. Biochemical analyses of recombinant protein variants lacking the membrane-anchored MHYT domain support NbdA being an active PDE. In contrast, MucR displayed both diguanylate cyclase and PDE activity in vitro, which seemed regulated in a growth-dependent manner in vivo. This is the first description of a PDE specifically involved in NO-induced biofilm dispersion in P. aeruginosa.

PAS Domain Residues and Prosthetic Group Involved in BdlA-Dependent Dispersion Response by Pseudomonas aeruginosa Biofilms

Biofilm dispersion by Pseudomonas aeruginosa in response to environmental cues is dependent on the cytoplasmic BdlA protein harboring two sensory PAS domains and a chemoreceptor domain, TarH. The closest known and previously characterized BdlA homolog is the flavin adenine dinucleotide (FAD)-binding Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Here, we made use of alanine replacement mutagenesis of the BdlA PAS domain residues previously demonstrated to be essential for aerotaxis in Aer to determine whether BdlA is a potential sensory protein. Five substitutions (D14A, N23A, W60A, I109A, and W182A) resulted in a null phenotype for dispersion. One protein, the BdlA protein with the G31A mutation (BdlA-G31A), transmitted a constant signal-on bias as it rendered P. aeruginosa biofilms hyperdispersive. The hyperdispersive phenotype correlated with increased interaction of BdlA-G31A with the phosphodiesterase DipA under biofilm growth conditions, resulting in increased phosphodiesterase activity and reduced biofilm biomass accumulation. We furthermore demonstrate that BdlA is a heme-binding protein. None of the BdlA protein variants analyzed led to a loss of the heme prosthetic group. The N-terminal PASa domain was identified as the heme-binding domain of BdlA, with BdlA-dependent nutrient-induced dispersion requiring the PASa domain. The findings suggest that BdlA plays a role in intracellular sensing of dispersion-inducing conditions and together with DipA forms a regulatory network that modulates an intracellular cyclic d-GMP (c-di-GMP) pool to enable dispersion.

The Phosphodiesterase DipA (PA5017) Is Essential for Pseudomonas aeruginosa Biofilm Dispersion

Although little is known regarding the mechanism of biofilm dispersion, it is becoming clear that this process coincides with alteration of cyclic di-GMP (c-di-GMP) levels. Here, we demonstrate that dispersion by Pseudomonas aeruginosa in response to sudden changes in nutrient concentrations resulted in increased phosphodiesterase activity and reduction of c-di-GMP levels compared to biofilm and planktonic cells. By screening mutants inactivated in genes encoding EAL domains for nutrient-induced dispersion, we identified in addition to the previously reported ΔrbdA mutant a second mutant, the ΔdipA strain (PA5017 [dispersion-induced phosphodiesterase A]), to be dispersion deficient in response to glutamate, nitric oxide, ammonium chloride, and mercury chloride. Using biochemical and in vivo studies, we show that DipA associates with the membrane and exhibits phosphodiesterase activity but no detectable diguanylate cyclase activity. Consistent with these data, a ΔdipA mutant exhibited reduced swarming motility, increased initial attachment, and polysaccharide production but only somewhat increased biofilm formation and c-di-GMP levels. DipA harbors an N-terminal GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain and two EAL motifs within or near the C-terminal EAL domain. Mutational analyses of the two EAL motifs of DipA suggest that both are important for the observed phosphodiesterase activity and dispersion, while the GAF domain modulated DipA function both in vivo and in vitro without being required for phosphodiesterase activity. Dispersion was found to require protein synthesis and resulted in increased dipA expression and reduction of c-di-GMP levels. We propose a role of DipA in enabling dispersion in P. aeruginosa biofilms.

Melatonin inhibits NO‐induced cGMP accumulation in porcine coronary arteries

Recent studies in our laboratory indicate that melatonin modulates vascular tone by interfering with nitric oxide (NO) signaling in coronary arteries. Here we tested the hypothesis that melatonin impairs NO‐induced vasorelaxation by inhibiting the NO‐induced increase in cGMP levels in coronary arteries. In organ bath studies, sodium nitroprusside (SNP) (10−9‐10−5 M) caused relaxation of isolated porcine coronary artery rings. Incubation of the tissues with melatonin (10−7 M) caused a rightward shift in the SNP concentration‐response curve. In the presence of the phosphodiesterase inhibitor, zaprinast (10−5 M), the inhibitory effect of melatonin on SNP‐induced vasorelaxation was abolished. Melatonin (10−7 M) had no effect on basal cGMP levels but markedly inhibited the SNP‐induced increase in cGMP. The effect of melatonin on cGMP accumulation was significantly diminished by incubation of the tissues with either zaprinast (10−5 M) or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). The expression of MT2‐receptors in coronary arteries was confirmed by immunoblot analysis. Taken together, these results suggest that the inhibitory effect of melatonin on NO signaling in coronary arteries is most likely due to impaired cGMP accumulation in response to NO. The inhibitory effect of melatonin is mediated by MT2‐receptors, perhaps via a mechanism that is coupled to increased phosphodiesterase activity.

Intravenous Tezosentan and Vardenafil Attenuate Acute Hypoxic Pulmonary Hypertension

Excessive hypoxic pulmonary hypertension imposes right ventricular strain by increasing afterload that may lead to right heart failure and death. Increased phosphodiesterase activity, as well as increased levels of endothelin-1, has been discussed as molecular mechanisms. We investigated the hemodynamic and intrapulmonary effects of the intravenous dual endothelin A and B receptor blocker tezosentan, and of the phosphodiesterase-5 (PDE-5) antagonist vardenafil in a pig model of acute normobaric hypoxic pulmonary hypertension. Eighteen 4-week-old ventilated white farm pigs were exposed to normobaric hypoxia (FiO2 12%) and randomly assigned to three groups (n = 6) in order to receive either intravenous tezosentan or vardenafil or to serve as control. Arterial alveolar oxygen differences were the same with both drugs. After 90 min of treatment, pulmonary artery pressure and vascular resistance were significantly lower in both treatment groups when compared to controls (p < 0.001). Cardiac index increased significantly with vardenafil alone (2.8 l x min(-1) x m2 +/- 0.7 to 4.2 l x min x m2 +/- 0.7, p = 0.0003). Intravenous tezosentan, as well as vardenafil equipotently attenuate acute hypoxic pulmonary hypertension without afflicting pulmonary gas exchange. However, cardiac index increases with vardenafil only.

Gamma-interferon production in atopic dermatitis shows differential modification by phosphodiesterase and prostaglandin inhibition.

Interferon-gamma (IFN-gamma) production by peripheral blood mononuclear leucocytes (MNL) is reduced in atopic dermatitis (AD) patients. This may be related to abnormalities in second messenger systems, and increased prostaglandin E2 (PGE2) release from monocytes. We compared the effects of manipulating the second messenger activity using the phosphodiesterase (PDE) inhibitor Ro 20-1724, dibutyryl cyclic adenosine monophosphate (cAMP), and cyclooxygenase inhibition of PGE2, on IFN-gamma production by cultured MNL from AD patients (n = 9) and normal controls (n = 10). Ficoll-Hypaque-separated MNL were cultured for 48 h with OKT3 stimulation, and cAMP, Ro 20-1724, or indomethacin. Supernatants were analysed for IFN-gamma by ELISA. Basal IFN-gamma was lower in AD patients, and the increase in IFN-gamma production with OKT3 was 6.5-fold greater in control subjects than patients with AD. Culture with indomethacin significantly enhanced OKT3-stimulated IFN-gamma production in both groups, whereas OKT3-stimulated IFN-gamma production was abolished with dibutyryl cAMP. IFN-gamma production was significantly lower with Ro 20-1742 in AD than in normal controls. We have shown reduced IFN-gamma release from unstimulated and stimulated MNL in AD patients compared with normal controls. The addition of indomethacin increased IFN-gamma production in both groups, although the increase was less in AD patients, suggesting an intrinsic cellular defect. IFN-gamma release from AD MNL was more sensitive to the inhibitory effects of PDE, and this may be due to increased PDE activity, or the hyperdynamic cAMP system present in atopics.

Induced Nitric Oxide Impairs Relaxation but Not Contraction in Endotoxin-Exposed Rat Pulmonary Arteries

Many patients with severe acute lung injury do not respond to nitric oxide (NO) inhalational therapy with alleviation of pulmonary arterial hypertension and hypoxemia, so this treatment remains controversial. MATERIALS AND METHODS.: We investigated in endotoxin-exposed Wistar rat pulmonary arteries whether endogenous NO alters contractile and relaxing responses, by electrochemical NO and isometric force measurements. Receptor-independent contraction was similar in control and endotoxin-exposed arteries, while thromboxane analogue (TxA)-dependent contraction was less in the latter. Neither non-selective NO synthase (NOS) inhibition by N(G)-nitro-l-arginine (l-NA) or selective inducible-NOS2 inhibition by aminoguanidine (AG) improved TxA-induced contraction in endotoxin-exposed arteries. Acetylcholine-induced relaxation was impaired in endotoxin-exposed pulmonary arteries, despite a comparable acetylcholine-induced NO release in control arteries. Additionally, NO solution-induced relaxation of endotoxin-exposed arteries was impaired, but could be improved by l-NA or AG. Application of a phosphodiesterase-insensitive cyclic guanosine monophosphate analogue induced similar relaxation in both control and endotoxin-exposed arteries. Endotoxin-associated NOS2-derived NO is thus associated with impaired NO-mediated relaxation, but does not underlie reduced receptor-mediated pulmonary contractile responses. An increased phosphodiesterase activity may underlie the former, so this route can be explored to replace or improve the effect of inhalational NO therapy in severe sepsis-induced acute lung injury in patients.

Guinea-pig lung adenylyl and guanylyl cyclase and PDE activities associated with airway hyper- and hypo-reactivity following LPS inhalation

The relationships between changes in in vivo airway reactivity and levels cyclicAMP and cyclicGMP were determined in guinea-pig lungs after exposure to inhaled lipopolysaccharide (LPS). After LPS (30 μg.ml −1, 1 h), guinea-pigs displayed in vivo airway hyperreactivity (AHR) at 1 h and hyporeactivity (AHOR) at 48 h, to inhaled (20 s) histamine (1 or 3 mM, respectively). Isoprenaline-stimulated cAMP or SNAP-stimulated cGMP were determined in the lungs isolated from guinea-pigs exposed to LPS inhalation to determine whether there was a relationship between AHR or AHOR and adenylyl/guanylyl cyclase and phosphodiesterase (PDE) activities. Assays were performed in the absence and presence of the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Levels of cAMP and cGMP in its presence indicated adenylyl and guanylyl cyclase activities, respectively. The difference between cAMP and cGMP levels, in the absence and presence of IBMX, reflected relevant PDE activity. In vivo AHR was associated with increased PDE activity towards cAMP and cGMP (67 and 278%, respectively) and also increased adenylyl (47%) and guanylyl (210%) cyclase activities. In vivo AHOR at 48 h after LPS inhalation was also associated with raised cyclase activity ( p < 0.05), whereas relevant PDE activity declined by 79 and 68%, compared with 48 h after vehicle. Although net stimulated cGMP levels increased during AHR and AHOR and net stimulated cAMP increased during AHOR, our index of PDE activity increased during AHR and decreased during AHOR. These results therefore support the rationale for the use of PDE-inhibitors in the treatment of respiratory diseases associated with AHR.

Diabetes-related changes in cAMP-dependent protein kinase activity and decrease in relaxation response in rat mesenteric artery

Using superior mesenteric artery rings isolated from age-matched controls and streptozotocin (STZ)-induced diabetic rats, we recently demonstrated that EDHF-type relaxation is impaired in STZ-induced diabetic rats, possibly due to a reduced action of cAMP via increased phosphodiesterase (PDE) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283-H291, 2003). Here, we investigated the activity and expression of cAMP-dependent protein kinase (PKA), an enzyme that is produced by a pleiotropic and plays key roles in the transduction of many external signals through the cAMP second messenger pathway and in cAMP-mediated vasorelaxation. The relaxation induced by cilostamide, a selective PDE3 inhibitor, was significantly weaker in superior mesenteric artery rings from STZ-induced diabetic rats than in those from age-matched controls. The relaxation responses to 8-bromo-cAMP (8Br-cAMP) and N6,O2-dibutyryl-adenosine-cAMP (db-cAMP), a cell-permeant cAMP analog, were also impaired in the STZ diabetic group. PKA activity in the db-cAMP-treated mesenteric artery was significantly lower in the STZ diabetic group. The expression levels of the mRNA and protein for PKA catalytic subunit Cat-alpha were significantly decreased in the STZ diabetic group, but those for PKA regulatory subunit isoform RII-beta were increased. We conclude that the abnormal vascular relaxation responsiveness seen in STZ-induced diabetic rats may be attributable not only to increased PDE activity but also to decreased PKA activity. Possibly, the decreased PKA activity may result from an imbalance between PKA catalytic and regulatory subunit expressions.

Relevant cAMP-specific phosphodiesterase isoforms in human pituitary: effect of Gs(alpha) mutations.

Both cAMP production by adenylyl cyclase and cAMP degradation by phosphodiesterases account for intracellular cAMP levels. We previously demonstrated an increased phosphodiesterase activity in GH-secreting adenomas bearing the gsp oncogene. Here we characterize both the activity and the expression of cAMP-specific phosphodiesterase genes in the human pituitary and in gsp+ and gsp- GH-secreting adenomas and analyze the impact of this intracellular feedback mechanism on the levels of cAMP-responsive element-binding protein phosphorylation. Normal pituitary and gsp- GH-secreting adenomas showed similar phosphodiesterase activities, and 7-fold higher levels were observed in gsp+ tumors. In these tumors the increased activity was mainly owing to isobutyl-methyl-xanthine-sensitive phosphodiesterase 4 and to isobutyl-methyl-xanthine-insensitive isoforms. By semiquantitative RT-PCR, all phosphodiesterase 4 transcripts were expressed in the normal and tumoral pituitary. However, the levels of phosphodiesterase 4C and 4D messenger RNAs were significantly higher in gsp+ than in gsp- GH-secreting adenomas and normal pituitary. Expression of the thyroid-specific isobutyl-methyl-xanthine-insensitive phosphodiesterase 8B was absent in the normal pituitary but detectable in almost all GH-secreting adenomas and higher in gsp+ (P < 0.02). Therefore, this study provides a characterization of phosphodiesterase expression in human pituitary and demonstrates a dramatic induction of the cAMP-specific phosphodiesterases 4C and phosphodiesterases 4D and phosphodiesterases 8B in gsp+ GH-secreting adenomas. Similar levels of cAMP-responsive element-binding protein phosphorylation were observed in gsp- and gsp+ GH-secreting adenomas; however, phosphodiesterase blockade caused an increase in cAMP-responsive element-binding protein phosphorylation that was significantly higher in gsp+ than in gsp- adenomas. Because cAMP-responsive element-binding protein represents the principal end point of the cAMP pathway, these results suggest that the enhanced phosphodiesterase activity may have a significant impact on the phenotypic expression of gsp mutations.

Genesis of pituitary adenomas: state of the art.

In recent years, remarkable progress has been made in the understanding of the pathogenesis of pituitary tumors. Pituitary tumors originate from the uncontrolled proliferation of a single transformed cell in which an initiating event has caused a gain of proliferative function. After the initiation, promoting factors cooperate in the clonal expansion. Common oncogenes, such as ras, are only exceptionally involved. The only activating mutations identified so far are gsp mutations causing the constitutive activation of cAMP pathway. However, gsp-positive adenomas are not associated to a more aggressive tumoral phenotype. The oncogenic potential of gsp mutations is limited by a more rapid degradation of the mutant Gs(alpha) with respect to the wild-type protein, and by a faster removal of cAMP due to increased phosphodiesterase activity. Estrogen-inducible gene sequences with transforming properties (pituitary tumor-transforming gene (PTTG)) have been identified in human pituitary tumors. Human pituitary tumor-transforming gene (hPTTG) is involved both in early pituitary tumorigenesis, as it causes in vitro and in vivo transformation acting as a transcription activator, and in tumor progression, as it regulates the production of basic fibroblast growth factor (bFGF), a potent activator of angiogenesis and mitogenesis. Moreover, a role of cyclin D1 in pituitary tumorigenesis is emerging. The allelic loss of loci for unknown oncosuppressor genes are currently under investigation, while an exceedingly limited role for menin gene and RB1 has been demonstrated for sporadic pituitary tumors. Abnormal methylation that predisposing toward genetic instability may favor the allelic loss or the reduced expression of oncosuppressor genes, is also an emerging field of investigation. Several promoting factors, including the excessive action of physiological stimulators, the defective action of inhibitors, the susceptibility to respond to inappropriate stimuli and the locally produced growth factors, help in tumor progression. The study of homeobox genes that intervene in pituitary cell differentiation may help in expanding our knowledge in pituitary tumor cell genealogy.

Desensitization of guanylyl cyclases in cultured human airway smooth-muscle cells.

We previously showed that cultured human airway smooth-muscle cells (HASMC) contain soluble and particulate guanylyl cyclases (GCs). We studied the desensitization of soluble and particulate GCs in HASMC. Homologous desensitization of soluble GC occurred after incubation with S-nitroso-N-acetyl pencillamine (SNAP). SNAP-dependent desensitization was blocked by hemoglobin, a nitric oxide (NO) scavenger, suggesting that it was due to NO release. Cross-desensitization between SNAP and sodium nitroprusside (SNP) and the lack of thiol reduction after SNAP or SNP treatment suggested that thiol depletion was not involved. Assays for soluble GC activity and experiments using protein synthesis inhibitors suggested that SNAP-dependent desensitization was due to reduced soluble GC. Homologous desensitization of particulate GC occurred after pretreatment with atrial natriuretic peptide (ANP) accompanied by reduced particulate GC activity. Recovery required protein synthesis, suggesting that it was due to reduction in particulate GC. Homologous desensitization to either SNAP or ANP was not altered by phosphodiesterase (PDE) inhibitors, suggesting that increased PDE activity was not involved. Cross-desensitization experiments using SNAP and ANP and experiments using zaprinast to elevate cyclic guanosine monophosphate showed no evidence of heterologous desensitization. Our results suggest that pretreatment of HASMC with SNAP or ANP causes homologous, but not heterologous, desensitization of soluble and particulate GCs, respectively.

Hypoxia-Mediated Modulation of Vascular Function—Implications for Organ Preservation and Thrombogenesis: Roger S. Mitchell Lecture

Hypoxia-Mediated Modulation of Vascular Function—Implications for Organ Preservation and Thrombogenesis: Roger S. Mitchell Lecture